DE3905505A1 - POLYMERS IGG DERIVATIVES FOR THE COMPENSATION OF STOER FACTORS IN IMMUNOASSAYS - Google Patents

Abstract

A method for the determination of a polyvalent substance which is capable of immunological binding in the presence of at least two reactants which are specific for the substance which is capable of immunological binding, in the presence of an aggregated substance with a concentration of 0.1 to 50 mu g/ml to compensate for interfering factors in human serum samples, and a reagent suitable for this purpose.

Description

The invention relates to an improved method for Bestim tion of an immunologically bindable polyvalent substance in the presence of at least two for the immunologically binding able substance specific reactants and in the presence an inhibitor for the compensation of interference factors in humans serum samples and a suitable reagent.

The sensitive determination of immunologically bindable Substances, such as polyvalent antigens (Peptides, proteins, polysaccharides, viruses, bacteria, specific cells) using two or more if several antibodies against different spatially Antigen determinants are known as immuno radiometric or immunoenzymometric sandwich assay (Two-site immunoassay). The most common is the implementation tion of this known determination method, wherein the determining antigen (sample) with a first antibody, the either in solid phase to a suitable support material such as Sepharose, agarose, plastic tubes and the like is or is homogeneous, such as biotinylated in Solution, and a certain amount of a second one or other labeled antibodies in the liquid phase is incubated. The specificity of the second and optionally Another antibody is preferably chosen so that it can be used against other determinants of the antigen to be determined than the first antibodies are directed to create a competition between the antibodies to the same binding sites on the be exclude voting antigen, as this testemp  would be impaired. Both the first, firmly phase-bound or biotinylated as well as the second or further, in solution, labeled antibodies are in the Added excess. The respective antigen can either over those fixed to the first antibody or those in solution remaining, not immunologically bound activity determined become. In the latter case, it is necessary to have a defined amount of labeled second antibody give.

Due to the required specificities, complete IgGs or their immunologically reactive fragments derived from monoclonal antibodies (MAbs) are used for the immunological sandwich assays (Fab, F (ab ') ₂ ). However, it is also possible to use immunoreactive components in these tests derived from polyclonal antibodies (PAHs).

Although in the described two-site immunoassays for the too specific analytes used specific antibodies who , human serum samples occur at a significant frequency that cause nonspecific reactions. These lead to wrong test results with corresponding serious Consequences for the therapeutic measures. Appearance of unspecific reactions is present in the sample Substances attributable to the same as the be determining analyte (polyvalent antigen) the specific Immunoglobulin reagents bind (interfering factors), however other attack points.

Usually, therefore, such immunoassays are preventive in the Excess non-specific immunoglobulins or immunoglobulin fragments (usually immune globulin G, IgG) from the same animal species of which the specific antibodies are added (G. M. Addison in Radioimmunoassay and Related Procedures in Medicine, Vol. 1, 131-147 (1974); EP-A 01 74 026). Since the use of specific monoclonal antibodies, which are usually  originated by the mouse, called in immunoassays called Anti-interference measure large amounts of nonspecific mouse IgG in the form of mouse berum, mouse ascites or isolated mouse Immunoglobulin (about 300-500 μg / ml; Clin. Chem. 32, 1491-1495 (1986)). For example, according to EP-A 01 74 026 Addition of approx. 30 μg / ml of relevant, non-specific mouse or rat IgG to immunoassays of the kind described only at certain sera and in special cases (negative samples) achieved a complete compensation of disturbances. The Providing mouse IgG in the required kilo-scale but with the currently available methods too economical interesting conditions not possible and also ethi critical views.

An essential measure for avoiding disturbances in immunoassays of the type mentioned is the use of Fab or F (ab ') ₂ fragments for at least one of the specific antibodies used in the immunoassay. This removes all confounding factors in the sample that are directed to Fc portions of IgG (rheumatoid factors, anti-Fc immunoglobulins such as IgM), their point of attack on one of the specific immune reagents and thus need not be compensated.

However, in some human serums, immunoassays continue to interfere despite the Fc-free specific antibody reagents used. These are due to substances in the serum, which are directed to Fab or F (ab ') ₂ . According to EP-B 00 83 869, however, such interference factors only recognize Fab regions if they are separated from the Fc part. These can be eliminated in appropriate immunoassays by the addition of native or crosslinked, for the antigen to be determined non-specific Fab or F (ab ') ₂ fragments (EP-B 00 83 869). Here, aggregated Fab or F (ab ') 2 fragments show a 2-3-fold higher suppression effect compared to the native components. By contrast, complete nonspecific IgGs according to EP-B 00 83 869, in both native and aggregated form, do not cause interference suppression in the immunoassays mentioned. The method also has the significant disadvantage that in this case - in addition to the specific for the antigen to be determined immunoglobulin reagents - significant amounts of high quality non-specific immunoglobulin reagents are needed, which brings considerable economic and ethical problems. Thereafter, for example, at least 100 μg / ml of the more efficiently proven aggregated nonspecific Fc-free immunoglobulin fragments are used for the suppression.

The invention is therefore based on the object of providing new possibilities for the compensation of unspecific reactions in immunoassays with Fc-containing and Fc-free specific antibody reagents as specific reactants, which results in complete IgGs and on Fab or F (ab '). ₂ fragments are reliably eliminated in human serums and, taking account of ethical considerations, allow the improved test systems to be used commercially.

This object is achieved by a method for the determination of an immunologically bindable substance in a biological fluid of a first species or by humans in the presence of at least two for the immuno logically binding substance specific reactants of a second species and in the presence of an inhibitor to Kompen sation of interference factors, characterized in that the Reaction mixture with 0.1-50 μg / ml for the immunological non-specific binding substance, monoclonal or polyclonal antibodies derived aggregates of a second and / or third species for the compensation of sturgeon factors is brought into contact.

As immunologically bindable substances in particular polyvalent antigens such as peptides, proteins, polysaccharides, viruses, bacteria and other specific cells or components of these come into consideration. Both polyclonal and monoclonal antibodies can be used as specific reactants for the immunologically bindable substance to be determined. IgGs and their immunoreactive constituents such as Fab or F (ab ') ₂ fragments are preferably used in combination. Particularly preferred is the use of two or possibly more specific reactants, of which at least one is a Fab or F (ab ') ₂ fragment and the other specific reactants are complete IgGs.

As for the immunologically bindable substance nicht specifi cal antibody aggregates to compensate for disorders preferably aggregates are used for the immunologically bindefä Hige substance non-specific IgGs, particularly preferably an IgG aggregate consisting of non-specific IgG and another macromolecule. Preference is given here to non-specific IgGs which originate from the same animal species as at least one of the specific reactants. As macro molecules here are, for example, Fab or F (ab ') ₂ fragments, ie Fc-free IgG fragments, which can originate from the mouse or another species of derived MAbs or PAHs, as well as proteins (eg. Albumin), polysaccharides (eg dextran) or other water-soluble polymers. In addition, heteropolymers formed from, for example, mouse IgG via bridging bovine IgG can be used to compensate for disorders. Especially preferred are aggregates consisting of an IgG molecule and a Fc-free IgG fragment, wherein both the IgGs and the Fc-free IgG fragments are from the same animal species as one of the specific reactants. Such IgG / Fab or F (ab ') ₂ polymers even show a factor of about 3 better suppression effect than pure IgG aggregates. Depending on the individual serum sample, 0.1-50 μg / ml of an IgG copolymer is sufficient for successful suppression, but preference is given to working at concentrations of 5 to 25 μg / ml and more preferably at concentrations of 5 to a maximum of 10 μg / ml since, in most cases, a much smaller addition of nonspecific IgG aggregates is fully effective.

According to a preferred embodiment of the invention, non-specific homopolymeric or heteropolymeric IgG aggregates or IgG / Fab or F (ab ') 2 aggregates having molecular weights of 320,000 daltons and more are used to compensate for interfering factors in human sera. The IgG aggregates contain IgG from the species from which at least one of the specific reactants originates and preferably belong to the same subclass as at least one of the specific reactants. Polymeric IgG preparations with molecular weights of 320,000 to 10 million daltons are preferably used in this case, the compensatory action against serum interference factors increasing with increasing molecular weight. Particularly preferred embodiments of the invention are two-site immunoassays with two or more specific mouse or rat MAK reactants, at least one reactant including a Fab or F (ab ') ₂ fragment and at least one of the other reactants Fc-containing IgG is in which compensating tion of interfering factors homopolymeric or heteropolymeric non-specific IgG aggregates or IgG / Fab or F (ab ') ₂ -Aggrega te be added with molecular weights greater than 320 000 daltons, wherein in The non-specific aggregates contain IgGs and Fab or F (ab ') ₂ fragments monoclonal and of the species and subclass are at least one of the specific reactants. It does not matter whether the nonspecific IgG or Fab fragment is produced via ascites, fermentation or genetic engineering by expression in microorganisms.

The method according to the invention for the compensation of disorderly polymerized nonspecific IgG Molecules or the nonspecific IgG- / Fab- or F (ab ') 2- Aggregates must be different species than the species the sample to be analyzed is derived. Usually be immunological determination methods for analysis of human serums with the help of specific ones, from the mouse itself deriving monoclonal antibodies or their immunreakti used fragments. The specific antibody reagents  However, tien may also be from another animal species come. The non-specific anti-interference added to the suppression Body aggregates can be used by the mouse or in the mouse Combination with mouse IgG or their Fab fragments of one other, from the first different animal species stam men. For example, mouse IgG / bovine IgG heteropolymers (see above) for suppression in immunoassays that are specific Antibody Reagent Use mouse MAbs or mouse IgGs be set.

The addition according to the invention of non-specific IgG aggregates or IgG / Fab or F (ab ') ₂ aggregates results in a factor of 20-1000 compared to native IgGs and in comparison to native or aggregated Fc-free IgG. Fragments a factor of 20-1500 or increased by 3 times effectiveness for the compensation of non-specific reactions in immunoassays with at least two for the immunogen-specific reactants to be determined, of which at least one of a Fab and a F (ab ') ₂ fragment derived reached. This is surprising since it was not foreseeable that IgG-containing nonspecific reactants in immunoassays in which Fc-free specific immunoglobulin reagents of monoclonal or polyclonal antibodies are involved, in the main a compensation of Fab or F (ab ') ₂ Fragments cause disordered interference. Furthermore, EP-B 00 83 869 as the closest prior art leads the skilled person rather to a contrary assumption. For it is expressly emphasized in this patent specification that both native and aggregated IgGs do not exhibit a disturbing effect in the immunological agglutination test mentioned there and that the agents added to the suppression as well as the agents added for the specific agglutination must be IgG-free.

Unspecific IgG can occur with itself or with antibody Fragments as well as with proteins, polysaccharides or others water-soluble macromolecules by heat, chemically or not covalently via bioaffinity interaction  be crosslinked according to methods known from the literature. In In any case, soluble in aqueous buffer solutions Aggregates. The chemical crosslinking can for example by homo- and heterobifunctional chemical linking arms (Linker), via proteins or activated dextran or through Self-crosslinking of the IgG molecules or their and / or others Fc-free fragments are made with carbodiimide. Besides, it is possible, the antibody monomers via Disulfidreduktion and Reoxidation or oxidation of their carbohydrate content with to crosslink themselves or a suitable macromolecule. As a chemical linker, for example, Bis (maleinimido) methyl ester, dimethyl suberimidate, disuccinimidyl suberate, Glutaric dialdehyde, N-succinimidyl-3- (2-pyridyldithio) propionate, N-5-azido-2-nitrobenzoylsuccinimide, N-succinimidyl (4-iodo acetyl) aminobenzoate or the combination of maleimido hexanoyl succinimidate and S-acetylmercaptobernstones acid anhydride or analogous compounds into consideration. The Production of activated dextran may be for example Aminodextran of a defined molecular weight with Maleinimidohexanoylsuccinimidat done which subsequently with mercaptoacetyl-derivatized non-specific IgG molecules is crosslinked. With bioaffiner Interaction is generally meant such bonds, as they z. B. in the pairs biotin / avidin (or streptavidin), Hapten / anti-hapten antibody, antigen / anti-antigen Antibody, ligand / binding protein (eg thyroxine / thyroxine binding protein), hormone / receptor. A suitable Embodiment results by at least two times Biotinylation of nonspecific IgG and addition of Streptavidin or by derivatization of IgG with at least two digoxigenin molecules and crosslinking with a monoclonal or polyclonal anti-digoxin antibody. Also suitable are aggregates obtained by reaction of Poly-human albumin with a monoclonal anti-human albumin Antibodies arise.

The aggregate crude mixtures obtained in each case can after Dialysis used directly, or for example by  Gel filtration in fractions of increasing molecular weight be separated and then directly to the compensation of interfering factors in immunoassays.

One of the added to the test mixture specific Reaktan th, preferably the Fc-containing IgG antibody is bound in the a conventional manner in the solid phase to a carrier mate rial such as agarose or plastic tubes and the like, or is - for example, connected with biotin - in the liquid phase in front. When a biotinylated first antibody is used, the complex of the antigen and a labeled second specific reactant is first formed, which is then bound in situ to a biotin-binding protein such as avidin or streptavidin-coated support material. However, other tests with first biotinylated antibody are also possible: The antigen reacts with the biotinylated MAb and is bound to a biotin-binding solid phase in situ or after a certain preincubation period and then brought together with the second specific reactant. Or it is possible that first the antigen reacts with the second specific reactant and then with the biotinylated antibody. The labeling of the second and optionally further specific reactants, which are preferably Fc-free IgG fragments, is carried out by coupling with an enzyme, a fluorescent, chemiluminezierenden or radioactive substance. Methods for labeling such antibody derivatives are known in the art, for example from E. Ischikawa et al., J. Immunoassay 4 (1983) 209-327, and need not be further explained here.

Another object of the invention are diagnostic Means for the determination of immunologically bindable polyva lenten substances in two-site immunoassays, which the described nonspecific antibody aggregates.  

The diagnostic agent contains besides two or given if several specific antibodies, one of which IgG molecule and at least one Fc-free IgG fragment is a suitable buffer system and the described unspe zifischen antibody aggregates and optionally further commonly used adjuvants, such as Reaction accelerators, detergents or stabilizers. When For example, suitable buffer systems may be 20-60 mM Phosphate buffer (pH 7.0) or a 50 mM HEPES / 100 mM NaCl Buffer system (pH 7.4) can be used. As Reaktionsbe For example, dextran sulfate or Polyethylene glycol 6000 to 40 000, as detergents z. B. Triton x-100, Tween 20 or Pluronic F 68 and as appropriate Stabilizers phenol, oxypyrion, chloroacetamide, merthiolate u. a. into consideration.

The diagnostic agent contains the nonspecific anti body aggregates with molecular weights of 320,000 daltons and more, preferably up to 10 million daltons, in one Concentration of 0.1 to 50 μg / ml, preferably in one Concentration of 5 to 25 μg / ml and more preferably in a concentration of 5 to 10 μg / ml.

The diagnostic agent may be in the form of a solution or a Trockenchemiereagents mounted on an absorbent carrier or in an open film.

The diagnostic agent according to the invention in the form of a Solution, optionally buffered to the desired pH, preferably contains all needed for the test Reagents. For durability reasons, it may be advantageous the reagents needed for the test to two or more Distributing solutions that are only at the actual Untersu be mixed. It is irrelevant whether the unspe isolated antibody aggregates separately in a suitable Buffer system and / or with one or / and two or more specific antibodies are presented.  

For the preparation of the diagnostic agent in the form of a Test strip becomes an absorbent carrier, preferably Filter paper, cellulose or synthetic fiber fleece with solutions of required, usually for the preparation of Teststrei Reagents used in volatile solvents like As water, methanol, ethanol or acetone impregnated. This can in an impregnation step he consequences. Often, however, it is appropriate to impregnation in to perform several steps, using solutions each one of the components of the diagno Sting agent included.

For the preparation of the diagnostic agent in the form of a Test strip can also serve an open film in which in addition to film formers and pigments, the specific antibodies or fragments, the nonspecific antibodies described per (fragment) aggregates, a suitable buffer system and other commonly used for diagnostic agents Additives are included.

The finished test papers and test films can be used as such be used or in a conventional manner to carrier foiled or preferably between plastics and feinmaschigen networks according to DE-PS 21 18 455 sealed and with the body fluid to be examined (eg Blood, plasma, serum).

The invention is suitable for the determination of all antigens with at least two antigenic determinants. Examples include thyrotropin (TSH), carcinoembryonic antigen (CEA), hepatitis B surface antigens (HBs), 1-fetoprotein (AFP), human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH ), β ₂ -microglobulin, prostatic acid phosphatase, prolactin, ferritin and insulin.

The following examples further illustrate the invention:  

Example 1 Preparation of monoclonal mouse IgG aggregate by Vernet tion with a homobifunctional reagent

Monoclonal MAK33 IgG (<95% pure, subclass composition K, γ 1, anti-creatine kinase specificity) is isolated from ascites fluid by ammonium sulfate precipitation and chromatography on DEAE ion exchangers (see A. Johnstone and R. Thorpe, Immunochemistry in practice, Blackwell Scientific Publications 1982, pages 44-45).

50 mg of IgG are dissolved in 3 ml of 0.025 M bicarbonate / carbonate buffer pH 9.5 solved. In this solution is pipetted 17 ul 12.5% pure Glutardialdehydlösung and incubated for 2 h at 25 ° C. to closing the solution is cooled in an ice bath and with 50 mM triethanolamine buffer adjusted to pH 8.2. This Solution is added 0.6 ml of freshly prepared sodium borohydride solution (8 mg sodium borohydride / 1 ml redistilled water) to and incubate for a further 2.5 h at 0 ° C. By 16 h dialysis at 0-4 ° C against 10 mM phosphate buffer pH 75, containing 0.2 M NaCl, Excess reagents are removed. The dialysate will concentrated by ultrafiltration to a volume of 1.25 ml. Part of this concentrate (raw mixture) is added directly to the Suppression used; 1.0 ml of it is passed through a gelfil column chromatographed with AcA22 filling (LKB). Column dimension 2 × 40 cm; Operating buffer 10 mM phosphate / l00 mM NaCl pH 7.5. The eluate of the column is equipped with a UV monitor assayed and fractionated at 280 nm for protein content. The Fractions of total protein-containing elution range are pooled into 4 pools of equal volume. By Calibration of the gel chromatography column with proteins known Molecular weight can give the pools the molecular weight 160,000-400,000, 400,000-1,000,000, 1-2 million and 2-10 million. After concentration of  Pools to a protein concentration of about 2 mg / ml the IgG aggregate solutions of different molecular weight areas are used for suppression.

Example 2 Preparation of monoclonal mouse IgG aggregate by Vernet tion with a heterobifunctional Reaqenz a) Preparation of MAK33-IgG-MH (Maleinimidohexanoyl-MAK33- IgG)

100 mg of MAK33 IgG are dissolved in 4 ml of 30 mM phosphate buffer pH 7.1 solved. To this solution is pipetted 20 μl (4 μmol) a 0.2 M solution of maleimidohexanoylsuccinimidate in Dimethyl sulfoxide. The reaction mixture is at 25 ° C for 1 h incubated for 16 h against 10 mM phosphate buffer pH 6.1, containing 50 mM NaCl, dialyzed at 0-4 ° C. You get 4.45 ml solution with 22 mg MAK33-IgG-MH / ml.

b) Preparation of MAK33 IgG SAMS (S-acetylmercaptosuccinyl) MAB33 IgG)

100 mg MAK33 IgG are dissolved in 4 ml 0.1 M phosphate buffer pH 8.0 solved. To this solution is pipetted 40 ul of 0.25 M Solution of S-acetyl-mercaptosuccinic anhydride in dime sulfoxide. The reaction mixture is incubated for 1 h at 25 ° C brewed, then 16 h against 10 mM phosphate buffer pH 6.1, containing 50 mM NaCl, dialyzed at 0-4 ° C. You get 4.45 ml with 21.8 mg MAK33 IgG SAMS / ml.

c) Crosslinking of MAK33 IgG MH with MAK33 IgG MS (Mercapto succinyl-MAB33-IgG)

Dilute 50 mg MAK33 IgG SAMS to a concentration of 15 mg / ml in 25 mM phosphate buffer pH 6.5, containing 2 mM  Ethylenediaminetetraacetate (Buffer A). Pipet to this solution 75 μl of a 1 M hydroxylamine solution are incubated 20 min at 25 ° C.

3 ml of this solution with 44 mg MAK33-IgG-MS are diluted with buffer A ad 6.0 ml. To this solution is added 4 ml of solution containing 88 mg of MAK33-IgG-MH. The mixture is incubated for 40 min at 25 ° C, then ceased to terminate the crosslinking reaction ad 2 mM with cysteine. After a further 30 minutes incubation at 25 ° C ad 5 mM N-methylmaleimide is added and kept at 25 ° C for 1 h. The reaction solution is dialyzed against 10 mM phosphate buffer pH 7.5 containing 0.2 M NaCl. The dialysate is concentrated by ultrafiltration to a protein concentration of 35 mg / ml and freed by low turbidity centrifugation. A typical molecular weight distribution for such a dialysate (Rohge mixed) of a MAK33-IgG aggregate, Fig. 1. The aggregate mixture can be used directly or after separation in molecular weight fraction (AcA22 chromatography as in Example 1) for filtering.

Example 3 Production of MAK33-IgG Aggregate via Crosslinking aminodextran a) Preparation of S-acetylthioacetyl-aminodextran

100 mg of aminodextran (Dextran T 40, Pharmacia, 28 Aminogrup pen / molecular weight 40,000) are dissolved in 4 ml of 30 mM phosphate buffer pH 7.1 dissolved. To this solution is pipetted 0.25 ml a 0.2M S-acetylthioacetyl succinimidate solution in Di sulfoxide. The reaction mixture is at 25 ° C for 1 h incubated, then against 10 mM phosphate buffer pH 6.1, containing 50 mM NaCl, dialyzed. Yield 90 mg S-acetylthioacetyl aminodextran in 4.5 ml solution.  

b) crosslinking of MAK33-IgG-MH with thioacetyl-aminodextran

2 ml solution with 20 mg / ml S-acetylthioacetyl-aminodextran are carefully adjusted to pH 6.5 with 0.1 M NaOH and Ethylenediaminetetraacetate ad 2 mM added. To this solution pipette 40 .mu.l of a 1 M hydroxylamine solution and Inku brews for 20 min at 25 ° C. Subsequently, the solution with 25 mM phosphate buffer pH 6.5 diluted to 6.8 ml and 7.2 ml a solution containing 158.4 mg MAK33 IgG MH (manufactured as Example 2a). After 30 min incubation at 25 ° C gives to the reaction mixture cysteine ad 2 mM and after 30 min additionally added N-methylmaleimide ad 5 mM. After a Another hour of incubation at 25 ° C, the polymer dialyzed against 10 mM phosphate buffer pH 7.5 / 50 mM NaCl. To Centrifuging a slight turbidity gives 19.5 ml a solution of MAK33 IgG dextran aggregate with a Protein content of 7.1 mg / ml.

Example 4 Production of MAK K33 IgG Aggregate via Crosslinking Bovine IgG a) Preparation of bovine IgG MH

100 mg polyclonal bovine IgG are analogous to Example 2a) reacted with 4 .mu.mol Maleinimidohexanoylsuccinimidat. from 4.45 ml solution with 22 mg bovine IgG-MH / ml.

b) Preparation of MAK33 SATA (S-acetylthioacetyl-MAK33- IgG)

100 mg of MAK33 IgG are dissolved in 4 ml of 30 mM phosphate buffer pH 7.1 solved. To this solution is pipetted 20 .mu.l (2 .mu.mol) of a 0.1 M solution of S-acetylthioacetylsuccinimide in dimethyl  sulfoxide. The reaction mixture is incubated for 1 h at 25 ° C, then 16 h at 0-4 ° C against 10 mM phosphate buffer pH 6.1 / 50 dialyzed in mM NaCl. 4.45 ml of 22 mg solution are obtained MAB33 IgG SATA / ml.

c) Crosslinking of thioacetyl MAK33 IgG with bovine IgG MH

50 mg MAK33 IgG SATA are mixed with 25 mM phosphate buffer pH 6.5, containing 2 mM ethylenediaminetetraacetate (Buffer A) to one Concentration of 15 mg / ml diluted. Pipet to this solution 75 μl of a 1 M hydroxylamine solution are incubated 20 min at 25 ° C.

3 ml of this solution with 44 mg of thioacetyl-MAK33-IgG are used 4 ml solution with 88 mg bovine IgG-MH and incubated for 25 min at 25 ° C. Cysteine ad 2 mM is then used to terminate the crosslinking and after 30 min incubation at 25 ° C iodoacetamide ad 5 mM added. After another hour incubation at 25 ° C is for 16 h at 0-4 ° C against 10 mM phosphate buffer pH 7.5 / 50 dialyzed in mM NaCl. Obtained after centrifugation of the dialysate 8.9 ml of MAK33 IgG bovine IgG aggregate with a protein concentration of 13 mg / ml.

example 5 Production of MAb33-IgG aggregate crosslinked via MAK33 Fab a) Preparation of MAK33 Fab SATA

200 mg MAK33 IgG are cleaved with papain to Fab / Fc and MAK33 Fab from the mixture by chromatography on DE52 Cellulose (Whatman) isolated (method see A. Johnstone and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications 1982, 52-53). Yield 95 mg MAK33-Fab as salt-free lyophilisate.  

50 mg of MAK33-Fab are dissolved in 2 ml of 30 mM phosphate buffer pH 7.1 solved. To this solution is pipetted 30 μl (3 μmol) a 0.1 M solution of S-acetylthioacetylsuccinimidate in Dimethyl sulfoxide. The reaction mixture is at 25 ° C for 1 h incubated for 16 h at 0-4 ° C against 10 mM phosphate buffer pH 6.1 / 50 mM NaCl / 2 mM ethylenediaminetetraacetate dialyzed. This gives 2.6 ml of solution with 18.5 mg MAK33-Fab-SATA / ml.

b) Crosslinking of MAK33 IgG MH with Thioacetyl MAK33 Fab

30 mg MAK33 Fab SATA are mixed with 25 mM phosphate buffer pH 6.5 diluted to a concentration of 15 mg / ml. To this Solution is pipetted 50 .mu.l of a 1 M hydroxylamine solution and incubated for 20 min at 25 ° C.

1.5 ml of this solution with 22 mg of thioacetyl MAK33 Fab with 55 mg MAK33-IgG-MH (prepared as 2a) and with dist. Diluted to a total volume of 10 ml. The The mixture is incubated for 35 min at 25 ° C and then for demolition crosslinking with cysteine ad 2 mM. After 30 min at 25 ° C is added N-methylmaleimide ad 5 mM and again 1 h incubated at 25 ° C. Subsequently, the reaction solution 16 h at 0-4 ° C against 10 mM phosphate buffer pH 7.5 / 0.1 M NaCl dialysed. After centrifugation, 12.8 ml of clear Solution with 5.3 mg MAK33 IgG Fab aggregate / ml.

Example 6 Production of MAK33-IgG Aggregate via Crosslinking Biotin / streptavidin a) Preparation of biotinylated MAK33 IgG

50 mg of MAK33-IgG in 2.5 ml of 0.1 M potassium phosphate buffer, pH 8.5 were admixed with 1.13 mg of biotinyl- ε- aminocaproic acid N-hydroxysuccinimide (Boehringer Mannheim GmbH in 50 μl of dimethyl sulfoxide (molar ratio IgG: biotin = 1: 7.5) and, after mixing, incubated for 90 min at 25 ° C. The biotinylated IgG was dialyzed overnight at 4 ° C. against 2 mM potassium phosphate buffer, pH 7.0 Yield: 45 mg MAK33-IgG-biotin in 6 , 4 ml.

b) Crosslinking with streptavidin

4 ml of the MAK33-IgG-biotin solution were spaced at 5 min with 3 portions of 1.5 mg streptavidin (Boehringer Mannheim GmbH) in 50 mM potassium phosphate buffer / 0.1 M sodium chloride and 20 min at 25 ° C. incubated. IgG molar ratio: streptavidin = 2.5: 1. The batch was concentrated by ultrafiltration to 2 ml trated and as in Example 1 on an AcA22 column Chromatograph. All fractions with molecular weight < 320,000 were united.

Yield: 21 mg MAK33 IgG streptavidin aggregate in 12 ml Solution with 17 mg IgG content.

example 7 Crosslinking of MAK anti-human albumin with human albumin

40 mg human albumin (Behringwerke, Marburg) in 1 ml 50 mM Potassium phosphate buffer, pH 7.0, was heated by heating for 3 hours 70 ° C aggregated. After cooling to 25 ° C, a proportion with 25 mg human albumin aggregate with 50 mM potassium phosphate buffer / Diluted 0.1 M NaCl, pH 7.0 ad 2.5 mg / ml and at intervals of 5 min with 4 portions of 2.5 mg MAK MI anti-human albumin (Gamma 1, Kappa) in 0.2 ml of 50 mM potassium phosphate buffer, pH 7.0 added. After 20 min incubation at 25 ° C was carried through Ultrafiltration concentrated to 2 ml and as above on a AcA gel column chromatographed. The proteinaceous Fractions of molecular weight <320,000 were combined.

Yield: 12.4 mg MAK MI-IgG albumin aggregate in 7.8 ml 8.86 mg IgG content.  

Example 8 Comparison of interference suppression of native MAK33 IgG and various containing MAK33-IgG aggregates in a CEA enzyme immunoassay two specific MAK reactants

The reagents from a test pack Enzymun® CEA (Boehringer Mannheim GmbH) were used. The test tubes contained in this test are coated with a monoclonal, CEA-specific mouse IgG (IgG 1, K). The enzyme labeled reactant is a monoclonal, CEA spec. Mouse Fab peroxidase conjugate; Subclass composition of the Fab is K, γ 1.

The various MAK33 IgG preparations were in increasing Concentration each added to the incubation buffer and the Test carried out with a human serum (P 43) in which noticed lend effective interference factors were detected. Table 1 shows the concentration of MAK33-IgG preparation (in μg Pro tein / ml) in the incubation buffer, which the disorder so far suppressed that the correct analyte content in the normal range (1-3.5 ng / ml) was found.  

Table 1

Relative suppression function of various MAK33-IgG preparations

Example 9 Suppressive effect of MAK-IgG aggregates that do not over covalent, bioaffine bonds were cross-linked in a CEA Enzymun test.

Reagents and test procedure see Example 8. The actual CEA content of the human serum sample used, determined by a reference method was 2.8-4.2 ng / ml. Table 2 shows the read on a calibration curve  apparent CEA concentrations for a human serum sample (# 108), which contains confounding factors at different IgG aggregate additions to the incubation buffer.

Table 2

Suppression of IgG aggregates with bioaffinity crosslinking

From the table it can be seen that both bioaffinity crosslinked IgG aggregates based on IgG amount by weight Clear much more effectively than uncrosslinked IgG: Factor <500 or <60. The quantities used were selected that only partial suppression resulted; among such Conditions can namely the relative suppression effect of various preparations are best estimated.  

Example 10 Dependence of the suppression function on the molecular weight of the MAB33 IgG aggregate

Reagents and execution for this trial are like described in Example 8. The MAK33 IgG preparations are prepared according to Example 2.

Table 3

Suppressive effect of MAK33-IgG aggregates as a function of molecular weight

example 11 Comparison of the suppression effect of MAK-IgG aggregates, herge represents different monoclonal antibodies

Test tubes from an Enzymun® TSH package (Boeh ringer Mannheim GmbH) used with a monoclonal Anti-TSH mouse IgG (IgGl, K) are coated. The remaining Reagents were as in Example 8 of the Enzymun CEA package taken. Test lead according to the instructions of this pack. In this test, there can be no signal through a Analyte content of a serum sample, because the two specifi reactants have different specificity. It han This is therefore just a model test with serum samples only results in a signal above the blank value, if confounding factors are included. The MAK IgG used for comparison Aggregates were prepared analogously to Example 2c from MAK33.  

Table 4

Comparison of IgG Aggregates of Different Monoclonal Antibodies in the Suppressor Action in a Model Test

Extinction for a serum sample without CEA and without interference factor: 0162

Result: The suppression effect of different monoclonal MAK IgG aggregates is the same within the limits of error.  

Example 12 Anti-interference effect of MAK33-IgG aggregate and monomeric MAK33-IgG in a test with biotinylated MAK IgG reactants

In this test, two monoclonal heptatitis-Oberflä chenantigen (HBsAg) -specific MAb IgGs in biotinylated Form used (biotinylation carried out according to T. V. Updyke and G.L. Nicolson in Methods in Enzymology, 121 (1986) 717-725). One of them, MAK6E7-IgG is of the subtype IgGl / K, the others, MAK5A10 IgG, are of the subtype IgG2a, K. As the enzyme labeled reactant was used MAK5A10-Fab-POD conjugate det.

Composition of the incubation buffer:

40 mM phosphate buffer pH 7.0
0.2 M sodium tartrate
0.5% (w / v) bovine serum albumin
0.1% (w / v) bovine IgG
0.5% (w / v) Pluronic F 68
0.01% (w / v) phenol
200 ng / ml MAK6E7 IgG (biotinylated)
25 ng / ml MAK5Al0 IgG (biotinylated)
200 mU / ml MAK5A10 Fab POD conjugate
Suppressor protein supplement see Table 5.

MAK33 IgG aggregate crude mixtures were prepared according to Bei play 2c.

For the test were 0.2 ml serum sample and 1 ml Inkuba tion buffer into a polystyrene tube containing streptavidin coated, pipetted. It was then incubated for 4 h at RT. Subsequently, each tube was filled with 3 x 1.5 ml tubing  water and 1 ml substrate solution (ABTS / peroxide) from Enzymun CEA test pack. After another Hour incubation at RT, the absorbance in the vice set substrate solution at 405 nm.

Table 5

Comparison of the anti-interference effect of MAK33-IgG preparations in a sandwich enzyme immunoassay with biotinylated MAK IgG reactants

From the finding that for serum No. 100 the same suppression on a signal of 0.125 is achieved with 200 μg / ml monomeric MAK33 IgG or 1 μg / ml MAK33 IgG aggregate follows that for this test IgG aggregate is 200 times more interference suppressive.

Example 13 Suppression effect of MAK33-IgG aggregate in a test with Dry chemistry reagent

The determination is carried out with dry chemical reagent in one One step assay for the double antibody solid phase sandwich Principle in rotor insert elements with a Zentrifugalanaly sator with the analysis described in DE-OS 34 25 008.5 equipment performed.

1. Preparation of the reagent solutions a) Buffer I

50 mmol / l potassium phosphate buffer, pH 6.0 prepared by Mixture of 50 mmol / l K₂HPO₄ solution and 50 mmol KH₂PO₄- Solution until the pH reaches 6.0.

b) Buffer II

Buffer II is prepared as buffer I with the difference that the pH is adjusted to 7.5 and pH that the buffer additionally 10 g / l bovine serum albumin and Contains 150 mmol / l NaCl.

c) Reactant R₁ solution, bondable with TSH

Reactant R₁ is a mouse anti-TSH molecular Antibodies used. The antibody containing this Ascites fluid is ad 1.8 M with ammonium sulfate  added. The precipitate is suspended in a buffer of 15 mM Sodium phosphate, pH 7.0 and 50 mM sodium chloride. The solution thus obtained becomes a passage over DEAE-cellulose subjected. The thus obtained, TSH-bondable Antibody is biotinylated (5 biotin / IgG) and with buffer II to a protein concentration of 1 mg / ml diluted.

d) Enzyme-labeled reactant R₂ solution

As a reactant R₂ also a monoclonal mouse anti-TSH antibody is used, but recognizes a different antigenic determinant as reactant R₁. The ascites fluid containing this antibody is purified as indicated under 1.3. The complete antibody is prepared in a known manner by the method of RR Porter, Biochem. J. 73, (1959), p. 119, cleaved to the Fab fragment. The resulting Fab fragments are coupled with β -galactosidase according to Ishikawa et al., J. of Immunoassay 4 (1983), pp. 209-327. The reactant R₂ solution is diluted in buffer II to a concentration of 500 mU / ml (measured with o-nitrophenyl- β- galactoside at 37 ° C).

e) interference-suppressing

MAK33 IgG polymer, prepared as in Example 2, is incorporated in Buffer II dissolved to a concentration of 1 mg / ml.

f) Avidin Solution

Avidin is expressed in buffer I at a protein concentration of Diluted 50 μg / ml.  

g) Substrate Solution

Chlorophenol red β- galactoside (prepared according to DE-OS 33 45 748) 5 mmol / l (3.05 g / l) HEPES 70 mmol / l (16.7 g / l) NaCl 154 mmol / l (9 g / l) Bovine Serum Albumin 0.3% (3 g / l) Tween 20 0.2% (2 g / l) pH (with NaOH) 7.25

2. Preparation of reagent carriers a) Reagent carrier 1 (without interference suppression protein)

40 μl of a solution containing per liter 100 mmol sodium phosphate pH 7.3 (37 ° C), 2 mmol magnesium chloride, 9 g sodium chloride, 5 g bovine serum albumin, 0.5 mg biotinylated mouse anti-TSH monoclonal antibody (Reactant R₁), 1000 U anti-TSH antibody (mouse) Fab fragment β- galactosidase conjugate (Reactant R₂ solution) (activity determined with ortho-nitrophenyl- β- D-galactoside at 37 ° C) is added to Non-woven material consisting of commercial polyester paper. It is then dried at room temperature. These webs are stored at 4 ° C and a relative humidity of 20% until used.

b) Reagent carrier 1 '(with interference suppression protein)

The preparation is carried out as for reagent carrier 1 up to the difference that in addition per liter of impregnation solution 0.01 g MAK33-IgG aggregate from Example 2c (crude mixture) are included.  

c) Reagent carrier 2

On a cellulose fleece is after the cyanogen bromide activation process (DE-OS 17 68 512) avidin (avidin solution) fixed, wherein per gram of fiber material 10 ug avidin Fixation are offered. By washing becomes uncoupled Antibody removed and the fleece gently Room temperature dried. The storage of the thus obtained Nonwovens follows analogously to reagent carrier 1.

3. Implementation of the provision

Determination with the aid of these two reagent carriers 1 and 2, or 1 'and 2', with the in the DE-OS 34 25 008.5 described apparatus for implementation analytical determinations.

This teaches a rotor insert for centrifugal analysis machines, consisting of a molded body, the one Sample application chamber containing a plurality of reagent fields communicating, each one with a certain reagent impregnated absorbent carrier material contain, at least one mixing valve chamber and a measuring chamber, which together a Probenflüssigkeitstransportweg form, from radially inward to radially outward, when the insert element on the rotor is attached, and at least one further chamber leads to the measurement guide and with the Probenflüssigkeitstransportweg at least partially identical.

Here, the Probenflüssigkeitstransportweg of a sample application chamber (P) via a filled with absorbent material containing buffer chamber (a) , a chamber (b) and a between the chambers (a) and (c) arranged first valve chamber ( VK 1 ) to one second valve chamber ( VK 2 ) and from there via the chamber (d) and via a collecting chamber ( AK) to the measuring chamber (K) . For receiving a further liquid, a substrate chamber (PK) designed as a pump chamber is provided, which is connected to the second valve chamber ( VK 2 ) via a metering device (DK) and capillary (cap) and an overflow chamber ( ÜK) (see FIG. 2).

To determine the extinction a (measurement signal included Interfering signal) become reagent carrier 1 and reagent carrier 2 used to determine the extinction b (specific Measuring signal without interference signal) are reagent carrier 1 'and 2 used.

Reagent carrier 1 or 1 'is placed on field C of the rotor insert element and reagent carrier 2 on field d. In this case, 40 μl of concentrated sample are pipetted directly onto the field a through an opening at the upper edge. 270 μl of substrate solution are pipetted into chamber (PK) . By means of a suitable centrifugation program in which high speeds alternate with standstill, sample and substrate solution are then conveyed in the direction of separation matrix and cuvette.

In the course of the program, the reactants R₁ and R₂ without or with suppression protein by the sample liquid eluted from field c and the homogeneous mixture subsequently reacted. On field d, the complexes bound via R₁ to avidin. The Transfer of the sample from field c to d is within very short time.

The substrate solution is divided into portions by the dosing chamber (DK) , the first of which serve to wash out excess, uncomplexed conjugate.

Disturbing factors that could cross-link R₂ be used of 10 μg suppression protein / ml test solution neutralized and also washed out (use of Reagent carrier 1 ').

The β- galactosidase activity bound to d via complex formation is proportional to the amount of TSH contained in the sample or to the sample blank value. This activity is determined with a further substrate portion, wherein the substrate is reacted in a 5-minute reaction to colored products. The color formed and the further color development / min in the liquid phase are measured in the cuvette at 576 nm.

Under these conditions the following results were obtained:

Table 6

The content of TSH in human serum 43 was determined with an enzyme TSH test from Boehringer Mannheim confirmed to be 1.4 μU / ml. This test contains as specific reactants one on tube coated MAK anti-TSH and a polyclonal sheep anti-TSH TSH-Fab-POD conjugate. The latter is inert against interfering factors in HS 43.

Claims (17)

1. A method for the determination of an immunologically bindable substance in a biological fluid of a first species or of humans in the presence of minde least two specific for the immunologically bindable substance reactants of a second species and in the presence of an inhibitor for the compensation of Störfak factors, characterized that the reaction mixture with 0.1-50 ug / ml for the immunologically bindable substance nonspecific, derived from monoclonal or polyclonal antibodies aggregates of a second and / or third species is brought into contact to compensate for confounding factors. 2. The method according to claim 1, characterized in that it is the immunologically bindable substance to a polyvalent antigen, in the specific reactants to antibodies, of which at least one is a Fab or F (ab ') ₂ fragment, and non-specific aggregates are aggregated IgGs. 3. The method according to claim 1 or 2, characterized that the reaction mixture with 5-25 ug / ml not specific IgG aggregates is brought into contact. 4. The method according to claim 1 or 2, characterized that the reaction mixture with 5-10 ug / ml not specific IgG aggregates is brought into contact. 5. The method according to any one of claims 2 to 4, characterized characterized in that the non-specific IgG aggregates consist of IgG and a second macromolecule.   6. The method according to claim 5, characterized in that it is not specific for the second macromolecule IgGs of a third species to nonspecific Fc-free IgG fragments, proteins, polysaccharides or another water-soluble polymer is. 7. The method according to any one of claims 1 to 6, characterized characterized in that the non-specific aggregates a Molecular weight of 320,000 daltons and more. 8. The method according to any one of claims 1 to 6, characterized characterized in that the non-specific aggregates a Molecular weight of 320,000 to 10 million daltons respectively. 9. The method according to any one of claims 1 to 8, characterized characterized in that the nonspecific aggregates by heat, chemical or non-covalent Networking are formed. 10. The method according to any one of claims 1 to 9, wherein minde at least one of the specific reactants with a markie connected to another substance and another to a matrix fixed or biotinylated, and either the fixed one or to a biotin-binding protein associated immuno logically bound or the immunologically unbound Activity is determined, characterized in that the non-specific IgG aggregates in solution in the reaction tion mixture are present during the assay. 11. Diagnostic agent for the determination of an immunological bindable substance in a biological fluid from a first species or from humans, containing at least two for the immunologically bindable sub punch specific reactants of a second species, one Inhibitor for the compensation of disturbing factors, a ge suitable buffer substance and optionally further commonly used excipients, characterized  draws that to compensate for interference 0.1-50 μg / ml for the immunologically bindable substance specific, monoclonal or polyclonal antibody pern derived aggregates of a second and / or third Animal species with molecular weights over 320 000 daltons inserted be set. 12. A diagnostic agent according to claim 11, characterized in that it is in the immunologically bindable substance to a polyvalent antigen, in the specifi rule reactants to antibodies, of which at least one Fab or F (ab ') ₂ fragment and non-specific aggregates are aggregated IgGs. 13. A diagnostic agent according to claim 11 or 12, characterized characterized in that as nonspecific IgG aggregates consisting of IgG and a second macromolecule of IgG Aggregates are used. 14. A diagnostic agent according to claim 13, characterized records that the second macromolecule is around non-specific IgGs of a third species, not to specific Fc-free IgG fragments, proteins, polysaccharides ride or other water-soluble polymer. 15. A diagnostic agent according to any one of claims 11 to 14, characterized in that two or more specific mouse or rat MAK reactants, of which at least one reactant is a Fab or F (ab ') ₂ fragment and minde least one the other reactants is an Fc-containing IgG, and homopolymeric or heteropolymeric non-specific IgG aggregates or IgG / Fab or F (ab ') ₂ aggregates having molecular weights greater than 320,000 daltons are added, wherein the in the Non-specific aggregates IgGs and Fab or F (ab ') ₂ fragments are mono clonal and of the species and subclass are at least as one of the specific reactants. 16. A diagnostic agent according to any one of claims 12 to 15, characterized in that the non-specific IgG Aggregates have a molecular weight of 320,000 to 10 Million daltons. 17. A diagnostic agent according to claim 12 to 16, characterized characterized in that as an additional aid Reaction accelerators, detergents and / or Stabilizers are used.